A thromboxane A2 synthetase inhibitor retards hypertensive rat diabetic nephropathy

European Journal of Pharmacology, 210 (1992) 163-172 (c) 1992 Elsevier Science Publishers B.V. All rights reserved 11014-2999/92/$05.01)

163

EJP 522116

A thromboxane A 2 synthetase inhibitor retards hypertensive rat diabetic nephropathy H i d e m i M a s u m u r a % Satoshi K u n i t a d a b, Kiyoshi Irie h, Shinichiro A s h i d a b and Y o u i c h i A b e

,l

'~Department of Pharmacology, Kagawa Medical School, Japan and ~' Research Institute, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan Received 17 September 1991, accepted 22 October 1991

Spontancously hypertcnsive rats (SHR) were injected with strcptozotocin (STZ-SHR) to induce diabetes. The effect of DP-I904, a thromboxane A z synthctase inhibitor, on diabetic nephropathy was then studied by administering it for 5 months (1 or 10 mg/kg). DP-1904 did not affect renal 6-keto prostaglandin (PG)F~,~ production in STZ-SHR, but markedly inhibited renal thromboxane (TX) B 2 production, so that the 6-keto PGF~JTXB 2 ratio was significantly increased (P < 0.05). STZ-SHR showed significant uracmia and proteinuria, plus increases in urinary 7-glutamyl-transpeptidase and urinary N-acetyl-/3-glucosaminidase. DP-1904 significantly decreased (P < 0.0l) the urinary changes. STZ-SHR also showed an increase in mesangial periodic acid-Schiff-positive substance and in relative renal weight, both of which were significantly inhibited by DP-1904 (P < 0.05). Thus, DP-1904 inhibited both TXB~ production and the progression of renal damage in STZ-SHR. Thromboxane A2; Prostacyclin; Streptozotocin-induced diabetes; Diabetic microangiopathy; Thromboxane synthetase inhibitors

1. I n t r o d u c t i o n

In the treatment of diabetes mellitus, control of the vascular complications has been increasingly recognized as being important to the long-term prognosis, and in particular the prevention or control of the progression of microangiopathy is vital. In recent years, accelerated thrombogenesis has come to be regarded as the mechanism underlying the development of the vascular complications which are characteristic of diabetes. There are many reports suggesting an important role for the stimulation of platelet activity (Kwaan et al., 1972; Lagarde et al., 1980; Butkus et al., 1982; Halushaka et al., 1981). It has also been suggested that the interaction between prostacyclin (PGI 2) (Moncada et al., 1976), which is produced in the vascular wall and is a potent vasodilator and inhibitor of platelet aggregation, and thromboxane (TX) A 2 (Hamberg et al., 1975), which has the opposite effects, contributes to the development of vascular lesions (Moncada and Vane, 1979). Many studies with animal models of diabetes have found reduced vascular wall PGI 2 production (Harrison et al., 1980; Karpen et al., 1982) and the promotion of TXA 2 synthesis (Gerrard et al., 1980). Butkus et al.

Correspondence to: H. Masumura, Department of Pharmacology, Kagawa Medical School, 1750-1 Ikedo, Miki-cbo, Kida-gun, Kagawa 761-07, Japan. Tel. 81.878.98 5111, fax 81.878.98 7109.

(1980) and Ziboh et al. (1979) have also reported the occurrence of increased platelet TXA2 synthesis. Furthermore, Harrison et al. (1980) have reported a reduction of PGI2 production in the renal cortex in diabetes mellitus, suggesting that an imbalance between PGI 2 and TXA 2 might also be responsible for the progression of glomerular microangiopathy. Taken together, these findings suggested to us that selective inhibition of TXA 2 production might prevent the development of diabetic microangiopathy. DP-1904 is a novel imidazole-type TXA 2 synthetase inhibitor. Previous studies have demonstrated that DP1904 produces a selective, potent and long-acting inhibition of TXA 2 synthesis and platelet aggregation (Irie et al., 1986; Kanao et al.; 1989; Tanaka et al., 1989), and that it has a protective effect against experimental myocardial infarction, cardiac anaphylaxis, asthma, acute renal failure and hydronephrosis (Toda et al., 1990; Shibano et al., 1986; Takami et al., in press; Masumura et al., 1991). It has also been suggested that DP-1904 prevents restenosis after percutaneous transluminal coronary angioplasty in patients with effort angina and myocardial infarction (Yabe et al., 1989). In the present study, we investigated the effect of DP-1904 on the development and progression of nephropathy in a rat model of streptozotocin (STZ)-induced diabetes. In humans, diabetic nephropathy is frequently complicated by hypertension, which is considered to be closely related to the progression of the nephropathy (Christlieb et al., 1981; Fuller, 1985). Cooper et al.

164

(1986) have reported that diabetic nephropathy is more likely to develop in spontaneously hypertensive rats (SHR) than in W i s t a r - K y o t o rats (WKY). Therefore, we induced diabetes mellitus in both strains of rats and compared the changes in prostanoid metabolism which occurred.

2. Materials and methods

2.1. Int~estigation of prostanoid metabolism in rats with streptozotocin-induced diabetes The animals used were 5-week-old male S H R and W K Y purchased from Charles River of Japan. STZ was injected via the tail vein at a dose of 50 m g / k g (dissolved in 0.1 M citrate buffer, pH 4.5) to induce diabetes mellitus, and citrate buffer (1 m l / k g ) alone was injected in a control group of rats. Two months after the injection of STZ, rats were anesthetized by intraperitoneal (i.p.) administration of pentobarbital (30 m g / k g ) . Whole blood was obtained from the carotid artery. Immediately after the blood had been obtained, samples were centrifuged for 10 rain at 3000 rpm at 4°C in a refrigerated centrifuge to separate the serum, which was then stored at - 2 0 ° C for the T X B 2, 6-keto prostaglandin ( P G ) F ~ and serum glucose measurements. Serum glucose levels were measured with a Hitachi Type 736 autoanalyzer (Tokyo, Japan). At the same time, the thoracic aorta and left kidney were dissected out and cut into 1 mm (kidney) or 2 mm (aorta) slices. Kidney slices were prepared from the same region of each kidney, and the medulla and cortex were used in equivalent proportions so that intergroup comparisons were considered to be valid. These tissue samples were then suspendeded in Tris buffer (500 raM, p H 7.5) and the 6-keto P G F ~ and TXB 2 levels in the supernatant were measured. In brief, aortic rings and kidney slices were washed with 4 ml of Tris buffer at 4 ° C, and then incuated in 1.5 ml of Tris buffer at 37 o C for either 5 min (aortic rings) or 10 min (kidney slices). The reaction was stopped by adding 1.5 ml of Tris buffer at 4 ° C and the test tubes were simultaneously cooled in ice. The supernatant was assayed. The 6-keto P G F ~ and TXB 2 levels of the supernatant and serum were measured by radioimmunoassay (Remuzzi et al., 1985). The protein content of the tissue samples was determined by the method of Ix)wry (Lowry et al., 1951).

2.2. Effect of DP-1904 on SHR diabetic nephropathy The animals used were 5-week-old male S H R purchased from Charles River of Japan. Diabetic rats were prepared in the manner described above. One week after the administration of STZ, blood was taken from

the tail vein without anesthesia, and the blood glucose level was measured by the glucose oxidase method. STZ-treated S H R (STZ-SHR) showing a blood glucose level of not less than 300 m g / d l were used in the following experiments and were allocated to one of three groups (Group l, control rats; Group II, rats given DP-1904 at a dose of 1 m g / k g per day; and G r o u p II1, rats given DP-1904 at 10 m g / k g per day). Each group consisted of seven or eight rats. Non-diabetic S H R were injected with citrate buffer alone (instead of STZ in citrate buffer) and were also divided into three groups, each consisting of eight or nine rats (Group I, control rats; G r o u p II, rats given DP-1904 at 1 m g / k g per day; and Group lI1, rats given DP-1904 at 10 m g / k g per day). DP-1904 was dissolved in distilled water, and a single daily dose of 1 or 10 m g / k g was administered orally at 10:00 a.m., by gavage, to non-diabetic S H R and S T Z - S H R for 5 months, beginning 1 week after the injection of STZ or the vehicle alone.

2.2.1. Parameters determined during DP-1904 administration 2.2.1.1. Blood pressure and heart rate Blood pressure and heart rate were measured using the indirect tail cuff method of Pfeffer et al. (1971). 2.2.1.2. Urinary protein excretion, y-glutamyl transpeptidase (y-GTP), and N-acetyl-C3-glucosaminidase (NABG) le~els A 24-h urine specimen was collected 4 months after the injection of S T Z or vehicle alone from all rats, and the total urinary protein content was measured by the method of Lowry (Lowry et al., 1951). Levels of y - G T P ( y - G T P Test Pack, Sankyo Pharmaceutical Co., Tokyo, Japan) and N A B G (Hasebe, 1968) activities were also determined. 2.2.1.3. Urinary TXB 2 Five-hour urine specimens were collected from all rats 3 months after the injection of STZ or vehicle alone, and the urinary TXB 2 level was determined by radioimmunoassay (Remuzzi et al., 1985). 2.2.2. Parameters determined at the time of death One the day after the final dose of DP-1904, the rats were anesthetized with an i.p. injection of pentobarbital (30 m g / k g ) . Both kidneys were removed after collection of blood from the carotid arteries of anesthetized rats and weighed. The right kidneys in all groups were fixed in formalin for the preparation of tissue samples without any particular perfusion and examined histologically by using standard methods. In addition, TXB 2 and 6-keto PGFI~ production was determined in all rats in the S T Z - S H R groups, using preparations of the thoracic aorta and left kidney as described above. 2,2.2.1. Serum biochemical parameters The following parameters were measured, using a Hitachi Type 736 autoanalyzer (Tokyo, Japan): glutamic-oxaloacetic

165

transaminase (GOT), glutamic-pyruric transaminase (GPT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), glucose (Glu), triglycerides (TG), and potassium (K +). 2.2.2.2. Renal histology After being fixed in Lillie's buffer in formalin, kidneys were dehydrated and de±atted, and then embedded in paraffin. Two 4-p.m sections were obtained from each kidney, stained by the periodic acid-Schiff (PAS) method, and examined by light microscopy. The number of glomeruli showing vascular poles was counted on each section, and one of four grades (severe ( + + ), moderate ( + ) , slight (_+) and normal ( - ) ) was assigned to each glomerulus to indicate the severity of the increase in mesangial matrix. The incidence of glomeruli with lesions is shown as a percentage of the total number of glomeruli with vascular poles. Percentages were averaged for each section and compared among the groups.

2.3. Statistical analysis Experimental results are expressed as the means _+ S.E.M. Statistical comparisons were done with a oneway layout analysis of variance and multiple comparison, with the level of significance set at P < 0.05.

2.4. Materials The following materials were used in these studies: DP-1904 (Daiichi Pharmaceutical Co., Tokyo, Japan), streptozotocin (Sigma Chemical Co., St. Louis, MO, USA), TXB2, anti-TXB 2 antibody, 6-keto PGFm, and anti-6-keto PGF~,~ antibody (Seragen Inc., Boston, MA, USA).

TABLE

3. Results

3.1. Prostanoid metabolism in the t,ascular wall and kidney in diabetic SHR and WKY Table 1 shows the effects of STZ administration on blood glucose levels and the production of TXB x and 6-keto PGFI, ~ in serum, the aortic wall, and the kidney in S H R and WKY. In both strains of rat, the STZtreated groups had significant hyperglycaemia, but blood glucose levels of S T Z - S H R were slightly higher than those of STZ-WKY, but the difference was not statistically significant. Whole blood TXB z and 6-keto P G F m levels were significantly higher in the non-diabetic S H R and S T Z - S H R groups than in the non-diabetic WKY and STZ-WKY groups. Both S T Z - S H R and S T Z - W K Y showed a decrease in whole blood T X B 2 and 6-keto P G F m levels, but the difference was not statistically significant. The TXB 2 and 6-keto PGF~,, levels in the vascular wall were markedly raised in non-diabetic SHR, and were markedly reduced by the administration of STZ. Renal TXB 2 and 6-keto PGFI, ' levels were also significantly higher in S H R than in WKY. Renal 6-keto P G F m levels were lower in STZS H R than in non-diabetic SHR, while renal T X B , levels were significantly higher. The renal 6-keto PGF~,~/TXB 2 ratio was significantly lower in STZSHR. These changes were not observed in WKY, and thus prostanoid metabolism in the kidney after STZ administration clearly differed between S H R and WKY.

3.2. Effect of DP-1904 on SHR diabetic nephropathy 3.2.1. Body weight and kidney wet weight Table 2 shows the body weight and the wet weights of both kidneys combined. The administration of either

1

Effect o f s t r e p t o z o t o c i n ( S T Z ) d i a b e t e s o n s e r u m g l u c o s e levels, s e r u m levels o f T X B 2 a n d 6 - k e t o P G F m , a n d o n T X B 2 a n d 6 - k e t o P G F I , ~ p r o d u c t i o n in t h e a o r t a a n d k i d n e y in W i s t a r - K y o t o r a t s ( W K Y ) a n d s p o n t a n e o u s l y h y p e r t e n s i v e r a t s ( S H R ) . R a t i o : 6 - k e | o PGFE, ~ / T X B 2. All v a l u e s a r e r e p r e s e n t e d as m e a n s _+ S.E.

N u m b e r of a n i m a l s S e r u m g l u c o s e levels SerumTXB 2 6-ketoPGF m Ratio AorticTXB 2 6-keto PGF m Ratio Renal TXB 2 6 - k e t o PGF~,~ Ratio

(mg/dl) (pg/ml) (ng/ml) (pg/mgproteinper5min) (ng/mg protein per 5 min) ( p g / m l p r o t e i n p e r 10 m i n ) ( p g / m g p r o t e i n p e r 10 m i n )

WKY

STZ-WKY

SHR

STZ-SHR

8 184 ± 9 75 + 1 0 1.0_+ 0.2 13 + 4 115 + 1 0 7 . 0 ± 1.0 61 ± 5 92 ± 12 556 +_64 6 _+ 2

8 351 ± 14 b 51 ± 8 0 . 6 + 0.1 12 ± 3 98 ± 1 1 4 . 5 ± 0.8 ~ 46 ± 5 ~ 95 ± 2 1 482 + 5 8 5 + 1

8 189 + 10 400 ± 6 4 b 5.4+ 1.2b 14 + 4 270 + 16 I' 14.2+ 2.2 b 53 ± 6 174 ± 23 '~ 953 + 102 " 5 + 1

8 402 ± 15 d 310 + 5 2 3 . 6 ± (I.2 12 ± 2 150 + 1 5 d 5 . 0 ± 1.2 d 33 + 4 ~" 232 + 2 1 " 5111 + 8 4 d 2 ± lC

" P < 0.05 vs. W K Y , h p < 0.01 vs. W K Y , c p < 0.05 vs. S H R , d p < 0.01 vs. S H R .

166 TABLE 2 Effect of DP-1904 on body weight and wet weight of both kidneys in spontaneously hypertensive rats (SHR) and streptozotocin-treated SHR (STZ-SHR). N: number of animals. Treatment

SHR Control DP-1904 DP-1904 STZ-SHR Control DP-1904 DP-1904

Dose (mg/kg)

N

Body weight

Kidney w e i g h t / 1 0 0 g body weight

1 10

8 8 9

394 + 4 392+6 385_+8

0.62 + 0.113 0.61 _+0.04 0.59+0.04 ~'

1 10

7 7 8

280+ 15 t, 339_+ 10 " 351 _+9 ~"

0.91 +0.04 t, 11.75+0.03 ' 0.70_+0.01 c

-

" P < 0.05 vs. SHR control, b p < 0.01 vs. SHR conlrol, " P < 0.01 vs. STZ-SHR control.

1 or 10 m g / k g of DP-1904 to non-diabetic S H R did not significantly affect body weight. S T Z - S H R showed a significant inhibition of body weight gain when compared with non-diabetic SHR. This inhibition was clearly reversed by DP-1904 at doses of 1 or 10 m g / k g , and this effect lasted throughout the treatment period. In non-diabetic SHR, there was no significant change in the relative kidney weight at a dose of 1 m g / k g of DP-1904, but there was a significant decrease at 10 m g / k g . In STZ-SHR, the relative kidney weight was significantly increased when compared with non-diabetic SHR, and this increase was significantly inhibited by administration of 1 m g / k g or 10 m g / k g of DP-1904. 3.2. 2. Histological findings The histological features of the kidneys are shown in fig. 1. In non-diabetic SHR, the degree of PAS-positivity of the mesangium was not affected by the administration of DP-1904. Control S T Z - S H R showed a significant increase in mesangial PAS-positive substance when compared with control SHR, and this change was

dose dependently inhibited by the administration of DP-1904, with a significant difference being seen at a dose of 10 m g / k g . 3.2.3. Blood pressure and heart rate The initial blood pressure for the control S H R was 118 _+ 3.2 mm Hg, and this increased to 203 _+ 3.3 mm Hg 3 weeks after the start of the experiment. It thereafter remained almost constant throughout the experimental period. DP-1904 inhibited the development of hypertension in S H R in a dose-dependent fashion, and this effect lasted throughout the treatment period. The blood pressure in control S T Z - S H R was considerably lower than in control SHR, and DP-1904 had no significant effect on blood pressure in S T Z - S H R (fig. 2). Heart rate was not noticeably influenced by DP-1904 in any of the groups of rats (data not shown). 3.2.4. Urinary protein, y-GTP, N A B G and TXB 2 let~els DP-1904 had no effect on proteinuria or on 7 - G T P and N A B G levels in SHR. Control S T Z - S H R showed a significant increase in urinary protein excretion as well as in y - G T P and N A B G activities compared with control SHR. These parameters were significantly lower in S T Z - S H R given DP-1904 than in control STZ-SHR, and the improvement was dose-dependent (fig. 3). Urinary TXB 2 levels in control S Y Z - S H R were higher than in control SHR, and DP-1904 dose dependently decreased urinary TXB 2 levels in both S H R and STZS H R (table 3). An initial increase in urinary output was seen in the diabetic group, but it became the same as that in the non-diabetic group after 3 and 4 months. No significant difference was noted between the urine output in the DP-1904 and control groups. 3.2.5. TXB 2 and 6-keto PGF1, let,els in the l~ascular wall and kidney DP-1904 significantly inhibited TXB 2 production in the vascular wall in S T Z - S H R in a dose-dependent

PAS-positive substance in the mesangial tufts

F'I++ u SHR

L

Control(n=8)

Li::~::~::~::i::?:~::iiii!iii~i!:~'l / i i i DP-1904 (n=8)~ . / L i ~ : : i : : ? I~:= : i i ~ i ? : i i i .... 2l ~ 2 ! i ~ l -,.

i

1 mg

D P- 1904 (n = 9) i~!~!!i!i~i~iii~iiii:.iiiiiii 10 mg

Control (n = 7) DP-1904 (n=7) SI-Z-SH R 1 mg I___DP-1904 (n=8) U

lore,

,,

...... I

~+

I/

g

•

~ I

I

I

I

I

20

40

60

80

l

i J

I 100 (%)

Fig. 1. Effect of DP-1904 on the glomerular histological changes in spontaneously hypertensive rats (SHR) and streptozotocin-treated SHR (STZ-SHR). ( ): number of animals, * * * P < (1.001, * P < 0.05.

167

1

1.

j.

2oo

-

~. 15o

--u

I,~31~------

<> 4~

•

//p,~,

Alcd/.

SHR DP-1904

1 mg/kg (n=81

SHR DP-1904 10 mg/kg ( n = 9 )

/ill

m

0

STZ-SHR control (n=7)

Zi

STZ-SHR DP-1904 1 mg/kg (n=7)

I:l

STZ-SHR DP-1904 10 mg/kg (n=8)

100

o

, 0

, -1

~

, 3

, 4

~

n

, 21

weeks

Fig. 2. Effect of DP-1904 on systolic blood pressure in spontaneously hypertensive rats (SHR) and streptozotocin-treated SHR (STZ-SHR). ( number of animals, * * P < 0.01, * P < 0.05 vs. SHR control.

fashion and tended to increase 6-keto PGF~<~ production. In the kidney, TXB 2 production was also noticeably reduced by DP-1904, but 6-keto PGF1, ~ was not influenced. As a result, the 6-keto P G F 1 J T X B 2 ratio increased significantly in DP-1904-treated rats (figs. 4 and 5).

TABLE 3 Effect of DP-1904 on urinary excretion of thromboxane B 2 (TXB 2) in spontaneously hypertensive rats (SHR) and streptozotocin-treated SHR (STZ-SHR). N: number of animals, b.w.: body weight. Tr eatment

Dose (mg/kg)

N

TXB 2 excretion ( p g / 5 h per 100 g b.w.)

1 10

8 8 9

380+ 38.3 171 _+ 23.6 i, 95+ 7.2 b

1 10

7 7 8

526+_132.0" 167+ 29.0 b 80+ 13.2 ~,

SHR Control DP-1904 DP-1904 STZ-SHR

Control DP-1904 DP-1904

3.2.6. Serum biochemical parameters

In non-diabetic SHR, 5 months of daily oral administration of DP-1904 did not cause any noticeable changes in the biochemical parameters investigated. The control STZ-SHR showed marked increases in GOT, GPT, ALP, BUN, Glu, TG and K + compared with the control SHR. DP-1904 treatment considerably

~' P < 0.10 vs. SHR control, b p < 0.01.

TABLE 4 Effect of DP-1904 on serum parameters in spontaneously hypertensive rats (SHR) and streptozotocin-treated SHR (STZ-SHR). Drugs were administered orally once daily for 5 months. N: number of animals, GOT: glutamic oxaloacetic transaminase, GPT: glutamic pyruric transaminase, ALP: alkaline phosphatase, BUN: blood urea nitrogen, GLU: glucose, TG: triglyceride, K+: potassium. All values are represented as the m e a n s + S . E , Tr eatm ent

Dose (mg/kg)

N

GOT (IU/I)

GPT (IU/I)

ALP (KAUI

1 10

8 8 9

131_+13.5 132+26.5 115+-12.7

66.9_+ 7.13 51.0+ 4.41 54.8+- 8.83

27.3+ 27.2+ 29.6+

1 10

7 7 8

164_+26.0 116+- 6.54 112+- 12.0

87.6_+ 14.2 61.0+- 4.78 ~ 54.9+- 5.38 "

55.7+ 15.7 b 29.4+ (J.92 'j 28.2+ 1.61 d

BUN (mg/dl)

G LU (mg/dl)

TG (mg/dl)

K+ (mEq/l)

21.7+0.51 22.1+1/.79 23.2+0.68

207+-10.5 193+ 6.88 210+13.4

34.5+ 6.11 33.1+ 4.07 25.8+ 2.19

6.48+0.34 6.11+_0.40 6.11+_0.41

26.4+ 1.41 ~' 24.4+ 1.00 :' 24.1+0.92

41/4+72.2 b 276+ 14.6 ~"~ 295+33.0

81.9+ 16.7 b 60.7_+ 13.9 35.6+ 3.59 d

8.23_+0.67 " 7.31 _+0.62 6.311_+0.32 c

SHR Control DP-1904 DP-1904

1.37 1.29 1.77

STZ-SHR Control DP-1904 DP-1904

" P < 0./15, h p < 0.01 vs. SHR control, c p < 0.05, ,l p < 0.01 vs. STZ-SHR control.

168

U-GPT

U-Protein

U-NABG 1.0

=fi

.d C~

o

2

g

~;i~!!;~. . . .

'~~i~ !~i~ili !

ii!iiii!i:i:i:i:!

e-

!l

cn ~ o

0.5

J;:

iiiii!i!!!il:+:+i

tN

!ii!iiii!!~ I :;:::i:;!

E

iii~

E ~I

i!il iii

!

O'

(8) (8) (9)

(7) (7) (8)

SHR

S'I-Z-SHR

0

:.:.:,:.1

-i

(7) (7) (8)

SHR

' - ' ] : Control

STZ-SHR

1 mg/kg

V--I: DP-1904

iii!

iiii!! !!liill

.... i

lipidic!!iiiiiiii] 0

~

(8) (8) (9)

(7) (7) (8)

SHR

S'I-Z-SHR

: DP-1904 10mg/kg

Fig. 3. Effect of DP-1904 on urinary protein (U-Protein), y-glutamyl transpeptidase (U-y-GTP), and N-acetyl-fi-glucosaminidasc activity ( U - N A B G ) in spontaneously hypertensive rats ( S H R ) and streptozotocin-treated S H R (STZ-SHR). ( ): n u m b e r of animals, ** P < 0.01, * P < 0.05.

6-keto PGF~

TXB 2

6-keto PGF~=/I-XB2 **

600

=_oo

_L

........ :.:.:.:.

•

E

=

~;~

E

~'

,* []

::i::i:::::::: .'.'.'."

:

::::: ':'"' "

[~]: Control (n=7)

i:ii:i:

300

. . . . . . . . . .

:?

0

i:i:i:i:

if!

~

m 150

.1.:.:,:

:.:.:.:.: 0

[--17

: DP-1904 1 mg/kg (n=7)

[]

:i:i:i:i: :':':':': : DP-1904 10 mg/kg (n=8)

* P
Fig. 4. Effect of DP-1904 on aortic TXB 2 and 6-keto PGF~,, production in streptozotocin-treated spontaneously hypertensive rats. ( of animals, * * P < 0.01, * P < 0.05 vs. control.

TXB2 300

6-keto PGF,~ !

1

): n u m b e r

6-keto PGFI=/TXB~ 10

/

._= E

!

iiiiiiii

iiiil

150 o.

E

E

o.

0

o []

: Control (n = 7)

[]

: DP-1904 1 mg/kg (n=7)

iiiiii!iii []

: DP-1904 10 mg/kg (n=8)

* P
Fig. 5. Effect of DP-1904 on renal TXB 2 and 6-keto PGFI, ~ production in streptozotocin-treated spontaneously hypertensive rats. ( of animals, * P < 0.05 vs. control.

): n u m b e r

169

reduced the elevation of GPT, ALP, BUN, TG, and K + in STZ-SHR (table 4).

4. Discussion

Diabetic nephropathy can be considered to be a diabetic glomerulosclerosis in the narrow sense, and is thought to develop as a secondary response to diabetic microangiopathy (Mauer et al., 1981). Abnormal production of TXA 2 and PGI 2 in the vascular wall is regarded as an important factor in the development and progression of diabetic microangiopathy (Gerrard et al., 1980; Somova et al., 1988). PG12 is produced in the vascular wall, where it both inhibits platelet aggregation and causes the relaxation of vascular smooth muscle (Moncada et al., 1976). In contrast, TXA 2 is produced by platelets and is an extremely potent promoter of platelet aggregation and vascular smooth muscle contraction (Hamberg et al., 1975). Christlieb et al. (1981) have reported that patients with diabetic nephropathy frequently also develop secondary hypertension, which in turn may be one of the factors that aggravates diabetic glomerular lesions. In animal experiments, Cooper et al. (1986) reported that diabetic SHR were more prone to develop proteinuria than diabetic WKY. Yoshida et al. (1986) found that when diabetes mellitus was induced in two-kidney Goldblatt hypertensive rats, renal lesions were likely to develop that included minimal thrombogenesis accompanied by thickening of the glomerular basement membrane and hyaline arteriolosclerosis. Accordingly, we first developed a diabetic nephropathy model using SHR and WKY to analyze metabolism in the vascular wall and kidney. SHR showed a significantly increased production of TXB 2 and 6-keto P G F ~ in the vascular wall and kidney in comparison with WKY. When STZ was administered to these animals to induce diabetes, the vascular wall production of TXB 2 and 6-keto PGFI, ~ was reduced, and in particular the production of 6-keto PGF~,~ was markedly reduced in SHR. It is well known that PGI 2 production in the arterial wall is reduced in diabetes mellitus, and the present results are consistent with those obtained previously (Harrison et al., 1980; Karpen et al., 1982). This change in PGI 2 metabolism is considered to be related to a reduction in the production of arachidonic acid, due to a decrease in insulin-dependent desaturase activity (Holman et al., 1983; Poisson et al., 1978), as well as to a change in prostacyclin synthetase activity (Gerrard et al., 1980). Beitz and Forster (1980) have suggested that an abnormal lipid metabolism can affect arachidonic acid metabolism, stating that high-density lipoprotein increased PGI 2 synthetase activity while low-density lipoprotein reduced it. In STZ-WKY, renal 6-keto PGF~,~ production tended to be reduced but no change

occurred in TXB 2 production. STZ-SHR however showed a significant decrease in renal 6-keto PGFI~~ production and a marked rise in TXB 2 production. Recently, it has been demonstrated that abnormal prostanoid metabolism is associated with various renal disorders, such as acute ischemic renal failure, hydronephrosis and acute glomerulonephritis, and changes in TXB 2 production are considered to be an important factor in the progression of these conditions (Masumura et al., 1991; Llanos et al., 1983; Saito et al., 1984). These reports suggest that the acceleration of renal TXB 2 production in STZ-SHR could explain why these rats are more liable to develop diabetic nephropathy than STZ-WKY. STZ-SHR showed a significant increase in proteinuria, increased urinary y - G T P and NABG levels, and an increase in PAS-positive substance in the renal mesangium. NABG is an enzyme originating from lysozyme in the epithelial cells of the proximal renal tubules (Hir, 1979), while y - G T P is present in high concentrations in the proximal renal tubular brush border membrane (Orlowski and Meister, 1963). Since both of these enzymes show a high urinary excretion in renal impairment, it is possible that they can be used as indices of renal damage (Harauchi and Yoshizaki, 1990). It has been reported that NABG in particular is increased by diabetic microangiopathy (Whiting, 1979). Thus, the results mentioned above clearly indicate the development of renal impairment and suggest that STZ-SHR are a good model of diabetic nephropathy. The daily administration of DP-1904 for 5 months significantly retarded the deterioration in the urinary parameters investigated and also tended to decrease the serum BUN level. In addition, the increase in mesangial PAS-positive substance was inhibited by DP-1904. In patients in the initial stage of diabetic nephropathy, an increase in renal volume has been observed (Mogensen and Andersen, 1973), which is considered to be due to increases in renal glomerular blood flow and capillary pressure. When this state persists, the morphological changes of diabetic nephropathy supervene. In STZSHR, the renal wet weight was significantly greater than in non-diabetic SHR, and this increase was significantly inhibited by the administration of DP-1904. This result may be associated with the finding that the increase in PAS-positive substance in the mesangium was suppressed by DP-1904. DP-1904 did not alter the production of 6-keto PGFI(~, but noticeably reversed the increase in renal TXB z production in STZ-SHR. The action of DP-1904 on TXB 2 formation at a dose of 10 m g / k g was clearly stronger than that of a dose of 1 mg/kg, and the improvement in parameters of renal function (urinary protein, y - G T P and NABG) was also dose-dependent. Therefore, the production of TXB 2 and renal damage appear to be related. Our findings

17(I

suggest that DP-1904 may have prevented the development of glomerular microangiopathy by improving the balance between PGI 2 and TXB 2 in the kidney. However, the BUN level improved only slightly. The changes in this p a r a m e t e r were not in complete agreement with those in urinary protein and TXB 2 levels. The reason for this is unclear at present. The antihypertensive effect of daily DP-1904 administration has previously been reported in S H R (Irie et al., 1986). Our results also show that DP-1904 significantly inhibited the progression of hypertension in young SHR, and this effect lasted throughout the administration period. DP-1904 may have exerted this effect by potently and selectively inhibiting T X A 2 production and thus improving the balance between PGI 2 and T X A 2. However, S T Z - S H R developed hypertension much later than S H R did and to a lesser degree. Christlieb (1973) have reported that diabetic patients with both hypertension and renal disorders have low plasma renin activity and low aldosterone levels, and similar results have also been reported in an animal study with diabetic rats (Tokumori et al., 1984). These findings suggest the possibility that a decrease in blood pressure in hypertensive diabetics could be brought about by a decrease in renin and aldosterone secretion. The hyperkalemia observed in our S T Z - S H R may reflect the suppression of aldosterone production in these animals. The hyporeninemic hypoaldosteronism associated with diabetes mellitus is thought to be mediated by either a reduced activity of the sympathetic nervous system due to autonomic neuropathy (Inagaki et al., 1976; Vandongen et at., 1973), or an increase in the circulating plasma volume resulting from an increased plasma osmotic pressure (Christlieb and Long, 1979). Although the vascular wall 6-keto P G F ~ J T X B 2 ratio was significantly lower in S T Z - S H R than in non-diabetic SHR, the blood pressure was lower in the former group of rats. This suggests that the effects of the above-mentioned factors on blood pressure in diabetes are greater than the effect of the vascular wall P G I 2 / T X A 2 balance. DP-1904 did not change the blood pressure of S T Z - S H R significantly. It improved hyperkalemia and hyperglycemia and inhibited the antihypertensive effect of S T Z in SHR, but it also suppressed T X A 2 synthesis and altered the P G I 2 / T X A 2 balance in the vascular wall, thus causing vasodilation. Accordingly, the effects of DP-1904 on hyperkalemia, hyperglycemia and renin production in S T Z - S H R may have cancelled out the influence of the changes in the P G I 2 / T X A 2 ratio and thus had little net effect on blood pressure. There was no correlation between the effects of DP-1904 on blood pressure and on urinary parameters, so its antihypertensive action cannot be attributable to an improvement in renal function. It has been reported that diabetic patients with microangiopathy have a marked acceleration of platelet

aggregation activity (Heath et al., 1971; Bensoussan et al., 1975; O'Malley et al., 1975; Matsuo and Ohki, 1977), and abnormal platelet aggregation has been proposed as a cause of diabetic microangiopathy. However, up to now there have been few clinical reports on the use of antiplatelet agents for diabetic microangiopathy. Zabel-Langhennig et al. (Zabel-Langhennig et al., 1982) reported a double-blind controlled trial of the administration of aspirin to 120 diabetics for 5 years, which showed that the drug was ineffective in halting the progression of diabetic angiopathy. It has also been reported that the administration of aspirin for 6 months failed to improve hyperplastic retinopathy (Matsuo, 1983). Since aspirin reduces the production of both TXB 2 and PG12 by inhibiting cyclooxygenase activity, these studies fail to support the role of PGI 2 in diabetic microangiopathy. Recently, Barnett et al. (1984) reported that the administration of a T X A 2 synthetase inhibitor to diabetic patients reduced proteinuria. Since DP-1904 inhibits platelet aggregation (Irie et al., 1987) and also inhibits T X A 2 synthetase activity (but not PGI2 production) in the kidney and vascular wall, this agent may prove to be useful in the prevention a n d / o r treatment of diabetic microangiopathy.

References Barnett, A.H., K. Wakelin, B.A. Leatherdale, J.R. Brinon, A. Polak, J. Bennett, M. Toop, D. Rowe and K. Dallinger, 1984, Specific thromboxane synthetase inhibition and albumin excretion rate in insulin-dependent diabetes, Lancet 1, 1322. Beitz, J. and W. Forster, 1980, Influence of human low density and high density lipoprotein cholesterol on the in vitro prostaglandin 12 synthetase activity, Biochim. Biophys. Acta 620, 352. Bensoussan, D., S. Levy Toledano, P. Passa, J. Caen and J. Caniver, 1975, Platelet hyperaggregation and increased plasma level of Von Willebrand factor in diabetics with retinopathy, Diabetologia 11, 3(/7. Butkus, A., V.A. Skrinska and O.P. Schumacher, 1980, Thromboxane production and platelet aggregation in diabetic subjects with clinical complications, Thromb. Res. 19, 211. Butkus, A., E.K. Shirey and O.P. Schumacher, 1982, Thromboxane biosynthesis in platelets of diabetic and coronary artery diseased patients, Artery 11,238. Christlieb, A.R., 1973, Diabetes and hypertensive vascular disease. Mechanisms and treatment, Am. J. Cardiol. 32, 592. Christlieb, A.R. and R. Long, 1979, Renin-angiotensin system in phlorhizin compared with alloxan diabetes in the rat, Diabetes 28, 106. Christlieb, A.R., J.H. Warram, A.S. Krolewski, E.J. Busick, O.M. Ganda, A.C. Aswal, J.S. Soeldner and R.F. Bradley, 1981, Hypertension: The major risk in juvenile-onset insulin-dependent diabetes, Diabetes 30 (Suppl. 2), 90. Cooper, M.E., T.J. Allen, G. Jerums and A.E. Doyle, 1986, Accelerated progression of diabetic nephropathy in the spontaneously hypertensive streptozotocin diabetic rat, Clin. Exp. Pharmacol. Physiol. (Australia) 13, 655. Fuller, J.H., 1985, Epidemiology of hypertension associated with diabetes mellitus. Hypertension 7 (Suppl. 2), 3.

171 Gerrard, J.M., M.J. Stuart, G.H. Rao, M.W. Steffes, S.M. Mauer, D.M. Brown and J.G. White, 1980, Alteration in the balance of prostaglandin and thromboxane synthesis in diabetic rats, J. Lab. Clin. Med. 95, 950. Halushka, P.V., R.C. Rogers, C.B. Loadholt and J.A. Colwell, 1981, Increased platelet thromboxane synthesis in diabetes mellitus, J. Lab. Clin. Med. 97, 87. Hamberg, M., J. Svensson and B. Samuelsson, 1975, Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides, Proc. Natl. Acad. Sci. U.S.A. 72, 2994. Harauchi, T. and T. Yoshizaki, 1991). A method for determining urinary enzyme activities as nephrotoxic indicators in rats, Jap. J. Pharmacol. 54, 205. Harrison, H.E., A.H. Reece and M. Johnson, 19811, Effect of insulin treatment on prostacyclin in experimental diabetes, Diabetologia 18, 65. Hasebe, K., 1968, Biochemical studies on synovial fluid. I. Mucopolysaccharase activities in synovial fluid of rheumatoid arthritis, Fukushima J. Med. Sci. 15, 35. Heath, H., W.D. Brigden, J.V. Canever, J. Pollock, P.R. Hunter, J. Kelsey and A. Bloom, 1971, Platelet adhesiveness and aggr.egation in relation to diabetic retinopathy, Diabetologia 7, 308. tlir, M.L., 1979, Quantitative distribution of lysosomal hydrolase in the rat nephron, Histochemistry 63, 245. Holman, R.T., S.B. Johnson, J.M. Gerrard, S.M. Mauer, S. Kupcho-Sandberg and D.M. Brown, 1983, Arachidonic acid deficiency in streptozotocin-induced diabetes, Proc. Natl. Acad. Sci. U.S.A. 81), 2375. Inagaki, K., Y. Suzuki, K. Onishi, T. Watanabe and S. Mitsui, 1976, Neuropathy in experimental diabetic rats (1). MCV and teased peripheral nerves in alloxan-induced diabetic rats, J. Jap. Diab. Soc. 19, 8(/8. irie, K., S. Kunitada, H. Masumura, H. Kubo, S. Ashida and A. Akashi, 1986, Cardiovascular effect of a new thromboxane A~ synthetase inhibitor, 6-(imidazolyl-methyl)-5,6,7,8-tetra-hydronaph-thalene-2-carboxylic acid HCI (DP-1904), 6th International Conference on Prostaglandins and Related Compounds, June 3-6, Florence, P. 453 (Abstract). Kanao, M., Y, Watanabe, Y. Kimura, J. Saegusa, K. Yamamoto, H. Kanno, N. Kanaya, H. Kubo, S. Asbida and F. lshikawa, 1989, Thromboxane A 2 synthetase inhibitors. 2. Syntheses and activities of tetrahydronapthalene and indan derivatives, J. Med. Chem. 32, 1326. Karpen, C.W., K.A. Pritchard, Jr., A.J. Merola and R.V. Panganamala, 1982, Alterations of the prostacyclin-thromboxane ratio in streptozotocin induced diabetic rats, Prostagl. Leukotr. Med. 8, 93. Kwaan, H.C., J.A. Colwell, S. Cruz, N. Suwanwela and J.G. Dobbie, 1972, Increased platelet aggregation in diabetes mellitus, J. Lab. Clin. Med. 80, 236. Lagarde, M., M. Burtin, P. Berciaud, M. Blanc, B. Velardo and M. Dechavanne, 1980, Increase of platelet thromboxane A 2 formation and of its plasmatic half-life in diabetes mellitus, Thromb. Res. 19, 823. Lianos, E.A., G.A. Andres and M.J. Dunn, 1983, Glomerular prostaglandin and thromboxane synthesis in rat nephrotoxic serum nephritis. Effects on renal hemodynamics, J. Clin. Invest. 72, 1439. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Masumura, H., S. Kunitada, K. lrie, S. Ashida and Y. Abe, 1991, A thromboxane A 2 synthase inhibitor, DP-1904, prevents rat renal injury, Eur. J. Pharmacol. 193, 321. Matsuo, T., 1983, Disease conditions from the viewpoint of platelet agglutinability: Evaluation of platelet agglutinability in diabetics and chronic hemodialysis patients, Jap. J. Clin. Pathol. 31, 1168.

Matsuo, T. and Y. Ohki, 1977, Classification of platelet aggregation patterns with two A D P solutions (the double-ADP method) and its clinical application to diabetes mellitus, Thromb. Res. 11,453. Mauer, S.M., M.W. Steffes and D.M. Brown, 1981, The kidney in diabetes, Am. J. Med. 70, 603. Mogensen, C.E. and M.J. Andersen, 1973, Increased kidney size and glomerular filtration rate in early juvenile diabetes, Diabetes (U.S.A.) 22, 706. Moncada, S. and J.R. Vane, 1979, Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls, New Engl. J. Med. 301/, 1142. Moncada, S., R. Gryglewski, S. Bunting and J.R. Vane, 1976, An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation, Nature 263, 663. O'Malley, B.C., J.D. Ward, W.R. Timperley, N.R. Poter and F.E. Preston, 1975, Platelet abnormalities in diabetic peripheral neuropathy, Lancet 2, 1274. Orlowski, M. and A. Meister, 1963, y-Glutamyl-p-nitroaniline: A new convenient substrate for determination and study of L- and D-y-glutamyl transpeptidase activities, Biochim. Biophys. Acta 73, 679. Pfeffer, J.M., M.A. Pfeffer and E.D. Froblich, 1971, Validity of an indirect tail-cuff method for determining systolic arterial pressure in unanesthetized normotensive and spontaneously hypertensive rats, J. Lab. Clin. Med. (U.S.A.) 78, 957. Poisson, J.P., P. Lemarchal, J.P. Blond, J. Lecerf and F. Mendy, 1978, Influence du diabete alloxanique sur la conversion des acides linoleique et gamma-linolenique [1 14C] en arachidonate chez le rat in vivo, Diabete Metab. 4, 39. Remuzzi, G., L. Imberti, M. Rossini, C. Morelli, C. Carminati, G.M. Cattaneo and T. Bertani, 1985, Increased glomerular thromboxane synthesis as a possible cause of proteinuria in experimental nephrosis, J. Clin. Invest. 75, 94. Saito, H., T. Ideura and J. Takeuchi, 1984, Effects of a selective thromboxane A 2 synthetase inhibitor in immune complex glomerulonephritis, Nephron 36, 38. Shibano, T., Y. Suzuki, M. Takami and A. Akashi, 1986, Effect of DP-1904, a new thromboxane A 2 synthetase inhibitor, on cardiac anaphylaxis in guinea pig isolated heart (Abstract), Jap. J. Pharmacol. 40, 2571. Somova, I., G. Dashev, V. Kamenov, G. Kirilov, A. Kirjakov, M. Doncheva and M. Vassileva, 1988, Streptozocin-induced diabetes in rat. 1. Influence of hypertension and myocardial infarction on the development of vascular complications, Meth. Find. Exp. Clin. Pharmacol. 10, 677. Takami, M., Y. Takata, K. Matsumoto, S. Ono and W. Tsukada, Effect of DP-1904, a new thromboxane A 2 synthetase inhibitor, on guinea pig experimental asthma, Advances in the Understanding and Treatment of Asthma 19911 Oct. in London, Proceeding (in press). Tanaka, M., K. Ono, T. Takegoshi, T. Shiozawa, T. Suzuki, S. Nii and H. Shibata, 1989, The pharmacokinetics and pharmacodynamics of a new thromboxane synthetase inhibitor, 6-(l-imidazolylmethyl)-5,6,7,8-tetrahydronapthalene-2-carboxylic acid (DP1904), in man after single oral administration, J. Pharm. Pharmacol. 41,680. Toda, 1., A. Nozaki, K. Kawakubo, Y. Murakawa, H. Inoue, N. Yoshimoto and T. Sugimoto, 1990, Effects of a thromboxane A 2 synthetase inhibitor on ventricular fibrillation threshold during coronary artery occlusion and reperfusion, Jap. Heart J. 31, 87. Tokumori, Y., K. Murakami, A. Kurahashi, S. Kuno; O. Mokuda, T. Ikeda, A. Takeda, M. Tominaga and H. Mashiba, 1984, Renin secretion in streptozotocin-induced diabetic rats, J. Jap. Diab. Soc. 27, 107. Vandongen, R., W.S. Peart and G.W. Boyd, 1973, Andrenergic stimulation of renin secretion in the isolated perfused rat kidney, Circ. Res. 32, 290.

172 Whiting, P.H., 1979, N-Acetyl-/3-D-glucosaminidase level and diabetic microangiopathy, Clin. Chim. Acta 97, 191. Yabe, Y., K. Okamoto, H. Oosawa, M. Miyairi, H. Noike, M. Aihara and T. Muramatsu, 1989, Does a thromboxane A 2 synthetase inhibitor prevent restenosis after PTCA? (Abstract), Circulation 80 (Suppl. II), 26//. Yoshida, M., T. Horikoshi, K. Shirado, K. Kanki and A. Koide, 1986, The effects of hypertension on development of the diabetic nephropathy: Study in the streptozotocin diabetic rats with two-

kidney Goldblatt hypertension, Kidney Dialysis 21, 908 (in Japanese). Zabel-Langhennig, R., B. Ruttmann, I. Schiele, W. Schafer and W. Aisch, 1982, 5 Jahrige Kontrollierte Therapiestudie zur Prophy[axe der Diabetischen Angiopathie mit dem Plattchenfunktionshemmer Azetylsalizylsaure, Z. Gesamte Inn. Med. 37, 661. Ziboh, V.A., H. Maruta, J. Lord, W.D. Cagle and W. Lucky, 1979, Increased biosynthesis of thromboxane A~ by diabetic platelets, Eur. J. Clin. Invest. 9, 223.